阴立爽洗剂制备工艺及质量标准的研究
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摘要
本课题研究的阴立爽洗剂是纯中药外用经验方,由川黄柏,苦参,蛇床子,花椒,鹤虱,百部,地肤子,白鲜皮,苦楝皮,冰片等药材组成,是参照《本草纲目》和《滇南本草》,在总结古今治疗妇科疾病经验上,精炼验方而成。具有清热燥湿,祛风止痒的功效,用于治疗妇科霉菌性、细菌性阴道炎等引起的阴痒、红肿、灼痛等症状。
阴立爽洗剂因受中国药典2000年版标准和当时研究水平的限制,多年的临床应用发现,阴立爽洗剂制备工艺水平和质量标准有待改进。为解决其存在的问题,本课题利用现代中药提取分离技术,采用正交试验法L9(34)优选阴立爽洗剂制备工艺,同时改进专属性强的鉴别项目和含量测定项目,从而提高质量控制水平,进一步保证临床用药的安全性和有效性。
总而言之,本研究是在优选阴立爽洗剂最佳制备工艺的基础上,同时对阴立爽洗剂质量标准进行研究。本课题的研究内容包括下列几个部分：
论文第一部分：
与阴立爽洗剂主治功能相关疾病阴道炎的文献研究。阴道炎是妇科临床常见疾病,白带增多是阴道炎在临床上最常见的症状。西医学认为阴道炎被细菌、寄生虫的侵入感染或阴道内菌样失调所致,目前多采用全身抗炎加局部抗炎治疗。而中医学认为阴道炎属于“带下”、“阴痒”范畴,是由肝脾胃功能失调所致。中医学着重于整体调节,对于阴道炎的治疗,无论是改善症状、局部调理,还是祛除病因、从本论治,均有其独特之处。阴立爽洗剂的治疗作用与中药抗炎作用密切有关,故综述中药影响炎症免疫反应的机制研究进展,并对阴立爽洗剂组方和组方药材进行研究分析。
论文第二部分：
阴立爽洗剂制备工艺的研究。对组方中蛇床子、花椒、鹤虱三味药材,采用水蒸汽蒸馏法提取其中的挥发油成分,并采用正交试验法L9(34),以挥发油提取率作为量化考察指标,对浸泡时间、加水倍数和提取时间等三个因素进行考察,优选挥发油最佳提取工艺；继而将余下的川黄柏、苦参、百部、苦楝皮、白鲜皮、地肤子等药材合并上述残渣,加水,冷浸,并进行水提醇沉,室温静置24小时,滤过,挥到无醇味(回收乙醇),所得提取液合并上述挥发油(加吐温-80混合),然后加入冰片和适量1%对羟基苯甲酸乙酯溶液。采用正交试验法L9(34),以高效液相色谱法测定盐酸小檗碱的含量作为量化考察指标,对加水倍数、煎煮时间、煎煮次数和醇沉浓度等四个因素进行考察,优选水提醇沉最佳提取工艺。结果表明,挥发油最佳提取工艺是A2B3C3,即加水倍数为10倍,浸泡1.5小时,提取6小时；水提醇沉最佳提取工艺是E1F2G2H3,即加水倍数为10倍,煎煮2次,每次2小时,醇沉浓度为70%。
论文第三部分：
阴立爽洗剂质量标准的研究。以盐酸小檗碱为对照品,以三氯甲烷-甲醇-浓氨(50：10：1)为展开剂,对其中川黄柏药材进行薄层色谱鉴别；以苦参碱为对照品,以甲苯-丙酮-甲醇(8：3：1)为展开剂,对其中苦参药材进行薄层色谱鉴别；以蛇床子素为对照品,甲苯-乙酸乙酯-正已烷(3：3：2)为展开剂,对其中蛇床子药材进行薄层色谱鉴别；以十八烷基硅烷键合硅胶为色谱柱,以乙腈-0.1%磷酸溶液(50：50)(每100ml加十二烷基磺酸钠O.1g)为流动相,测定波长为265nm,采用高效液相色谱法测定阴立爽洗剂中盐酸小檗碱的含量。在薄层色谱中,阴立爽洗剂及川黄柏、苦参、蛇床子的对照药材与其对照品色谱相应的位置上,显相同颜色的斑点,阴性对照溶液无干扰。以各对照品检测浓度(X)为横坐标对其峰面积(Y)为纵坐标进行线性回归,回归方程：Y=4342774X+7252,r=1.0000,盐酸小檗碱线性范围为(0.0908-2.2700)μg,平均加样回收率99.8%,SD与RSD均为1.2%(n＝6)。
The Chinese herbal preparation Yinlishuang lotion, which is prepared by The Guangdong Provincial Corps Hospital Chinese People's Armed Police Forces, is consist of Phellodendron chinense cortex, Sophora flavescens radix, Cnidii fructus, Zanthoxyli pericarpium, Carpesii fructus, Stemonae radix, Kochiae fructus, Meliae cortex, Dictamni cortex, Borneolum syntheticum. It has the effect of clear away heat, dry dampness and wind-dispelling and itching-arresting. Yinlishuang lotion is used for negative itching、red swelling、causalgia, which causes by the vaginitis of mycotic and bacterium in the department of gynecology.
Since the limitations of study and the standard of Chinese Pharmacopoeia 2000 about Yinlishuang lotion, and it discovers that the Yinlishuang lotion need have had an improvement in the preparation technology and quality standard since several years of clinical application. To solve these problems, orthogonal experiment design L9 (34) is used to optimize preparation technology by the using of modem medicine extraction and separation technology. At the same time, we add the strong specificity identifications and the content of determination, so that we can enhance the level of quality control,and control the quality of Yinlishuang lotion.
Altogether, the research is on the foundation of optimizing the best preparation technology of Yinlishuang Lotion, also to carry on a research of establishing the quality standard for Yinlishuang lotion. The contents, method and conclusion of this research include a few of the aspects as follows:
The first part of this paper is the literature research of the related disease vaginitis, which Yinlishuang lotion cures. The vaginitis is a familiar clinical gynecology diseases, leucorrhea to increase is the most familiar symptom of the vaginitis on clinic. The appearance of vaginitis is irruptive infection, which is caused by bacterium or arasite or dysbacteriosis in vagina in western medicine. Currently, more adopt the therapy of systemic anti-inflammatory and local anti-inflammatory. The traditional chinese medicine learns to think vaginitis,which induced by the dysfunction of liver and spleen and stomach funtcionbelong to "leukorrhea" and"pruritus vulvae" category. The traditional chinese medicine learns to put great emphasis on whole regulate for the therapeutics of vaginitis. regardless improving symptom and monopathy pathology, still keeps dispeling in addition to the cause of disease cures from the theory of the traditional chinese medicine,so it has its special place. The therapeutic effects of Yinlishuang lotion has something to do with the anti-inflammatory effect of Chinese herbal medicine closely, so the past overview Chinese herbal medicine influences the mechanism research of the disease vaginitis immunity reaction progress, Chinese herbal medicine,which influences on the research progress of the inflammatory immunological reactions'mechanism,is summarized, and the composing prescriptions and prescription drugs of Yinlishuang lotion is carring on studying analysis.
The second part of this dissertation concentrated on the study on the quality standard of Yinlishuang lotion. By orthogonal experiment design L9(34),with extractive yield of volatile oil as an index, the optimal oil extraction condition was studied. Three factors were selected including water consumption, soaking time and extracting time. By orthogonal experiment design L9(34), with the content of berberine hydrochloride determined by HPLC as a quanlitative index, the optimal water extracting alcohol precipitating was studied. Four factors were selected including water consumption, extracting times, extracting time and the concentration of alcohol-sediment. The results have showed that the oil extraction condition is adding 10 times water, soaking for 1.5 hours and extracting for 6 hours. And the best water-extraction condition:boiled twice with 10 times water,2 hours for each time and 70% alcohol-sediment.
The last part of this dissertation concentrated on the study on the quality standard of Yinlishuang lotion. Berberine hydrochloride was used as reference substance and trichloromethane-methanol-concentrated ammonia solution(50:10:1) as developer to identify Phellodendron chinense by TLC;Matrine as reference substance and methylbenzene-acetone- methanol(8:3:1) as developer to identify Radix sophorae flavescentis by TLC; Osthole as reference substance and methylbenzene-ethyl acetate- n-hexane (3:3:2) as developer to identify Fructus cnidii by TLC; HPLC was used to determine Berberine hydrochloride in phellodendron chinense,the column was ODS-C18 colunm, the mobile phase was Methyl Cyanides-0.1%phosphoric acid (50:50) (add sodium dodecyl sulfate 0.1g per 100 ml),the detection wavelength was 265mn. The results showed that, Yinlishuang Lotion and the control medicinal material of Phellodendron chinense、Radix sophorae flavescentis and Fructus cnidii with respectively check against an article and check against the position department that the reference substance, respectively same spot by TLC-identification, and the negative control solution have noninterference. The regression equation is Y= 4342774X+7252, r=1.0000 for berberine hydrochloride, and the linear range was (0.0908-2.2700)μg, the average recovery was99.8%,SD and RSD are 1.2%(n=6).
阴立爽洗剂因受中国药典2000年版标准和当时研究水平的限制,多年的临床应用发现,阴立爽洗剂制备工艺水平和质量标准有待改进。为解决其存在的问题,本课题利用现代中药提取分离技术,采用正交试验法L9(34)优选阴立爽洗剂制备工艺,同时改进专属性强的鉴别项目和含量测定项目,从而提高质量控制水平,进一步保证临床用药的安全性和有效性。
总而言之,本研究是在优选阴立爽洗剂最佳制备工艺的基础上,同时对阴立爽洗剂质量标准进行研究。本课题的研究内容包括下列几个部分：
论文第一部分：
与阴立爽洗剂主治功能相关疾病阴道炎的文献研究。阴道炎是妇科临床常见疾病,白带增多是阴道炎在临床上最常见的症状。西医学认为阴道炎被细菌、寄生虫的侵入感染或阴道内菌样失调所致,目前多采用全身抗炎加局部抗炎治疗。而中医学认为阴道炎属于“带下”、“阴痒”范畴,是由肝脾胃功能失调所致。中医学着重于整体调节,对于阴道炎的治疗,无论是改善症状、局部调理,还是祛除病因、从本论治,均有其独特之处。阴立爽洗剂的治疗作用与中药抗炎作用密切有关,故综述中药影响炎症免疫反应的机制研究进展,并对阴立爽洗剂组方和组方药材进行研究分析。
论文第二部分：
阴立爽洗剂制备工艺的研究。对组方中蛇床子、花椒、鹤虱三味药材,采用水蒸汽蒸馏法提取其中的挥发油成分,并采用正交试验法L9(34),以挥发油提取率作为量化考察指标,对浸泡时间、加水倍数和提取时间等三个因素进行考察,优选挥发油最佳提取工艺；继而将余下的川黄柏、苦参、百部、苦楝皮、白鲜皮、地肤子等药材合并上述残渣,加水,冷浸,并进行水提醇沉,室温静置24小时,滤过,挥到无醇味(回收乙醇),所得提取液合并上述挥发油(加吐温-80混合),然后加入冰片和适量1%对羟基苯甲酸乙酯溶液。采用正交试验法L9(34),以高效液相色谱法测定盐酸小檗碱的含量作为量化考察指标,对加水倍数、煎煮时间、煎煮次数和醇沉浓度等四个因素进行考察,优选水提醇沉最佳提取工艺。结果表明,挥发油最佳提取工艺是A2B3C3,即加水倍数为10倍,浸泡1.5小时,提取6小时；水提醇沉最佳提取工艺是E1F2G2H3,即加水倍数为10倍,煎煮2次,每次2小时,醇沉浓度为70%。
论文第三部分：
阴立爽洗剂质量标准的研究。以盐酸小檗碱为对照品,以三氯甲烷-甲醇-浓氨(50：10：1)为展开剂,对其中川黄柏药材进行薄层色谱鉴别；以苦参碱为对照品,以甲苯-丙酮-甲醇(8：3：1)为展开剂,对其中苦参药材进行薄层色谱鉴别；以蛇床子素为对照品,甲苯-乙酸乙酯-正已烷(3：3：2)为展开剂,对其中蛇床子药材进行薄层色谱鉴别；以十八烷基硅烷键合硅胶为色谱柱,以乙腈-0.1%磷酸溶液(50：50)(每100ml加十二烷基磺酸钠O.1g)为流动相,测定波长为265nm,采用高效液相色谱法测定阴立爽洗剂中盐酸小檗碱的含量。在薄层色谱中,阴立爽洗剂及川黄柏、苦参、蛇床子的对照药材与其对照品色谱相应的位置上,显相同颜色的斑点,阴性对照溶液无干扰。以各对照品检测浓度(X)为横坐标对其峰面积(Y)为纵坐标进行线性回归,回归方程：Y=4342774X+7252,r=1.0000,盐酸小檗碱线性范围为(0.0908-2.2700)μg,平均加样回收率99.8%,SD与RSD均为1.2%(n＝6)。
The Chinese herbal preparation Yinlishuang lotion, which is prepared by The Guangdong Provincial Corps Hospital Chinese People's Armed Police Forces, is consist of Phellodendron chinense cortex, Sophora flavescens radix, Cnidii fructus, Zanthoxyli pericarpium, Carpesii fructus, Stemonae radix, Kochiae fructus, Meliae cortex, Dictamni cortex, Borneolum syntheticum. It has the effect of clear away heat, dry dampness and wind-dispelling and itching-arresting. Yinlishuang lotion is used for negative itching、red swelling、causalgia, which causes by the vaginitis of mycotic and bacterium in the department of gynecology.
Since the limitations of study and the standard of Chinese Pharmacopoeia 2000 about Yinlishuang lotion, and it discovers that the Yinlishuang lotion need have had an improvement in the preparation technology and quality standard since several years of clinical application. To solve these problems, orthogonal experiment design L9 (34) is used to optimize preparation technology by the using of modem medicine extraction and separation technology. At the same time, we add the strong specificity identifications and the content of determination, so that we can enhance the level of quality control,and control the quality of Yinlishuang lotion.
Altogether, the research is on the foundation of optimizing the best preparation technology of Yinlishuang Lotion, also to carry on a research of establishing the quality standard for Yinlishuang lotion. The contents, method and conclusion of this research include a few of the aspects as follows:
The first part of this paper is the literature research of the related disease vaginitis, which Yinlishuang lotion cures. The vaginitis is a familiar clinical gynecology diseases, leucorrhea to increase is the most familiar symptom of the vaginitis on clinic. The appearance of vaginitis is irruptive infection, which is caused by bacterium or arasite or dysbacteriosis in vagina in western medicine. Currently, more adopt the therapy of systemic anti-inflammatory and local anti-inflammatory. The traditional chinese medicine learns to think vaginitis,which induced by the dysfunction of liver and spleen and stomach funtcionbelong to "leukorrhea" and"pruritus vulvae" category. The traditional chinese medicine learns to put great emphasis on whole regulate for the therapeutics of vaginitis. regardless improving symptom and monopathy pathology, still keeps dispeling in addition to the cause of disease cures from the theory of the traditional chinese medicine,so it has its special place. The therapeutic effects of Yinlishuang lotion has something to do with the anti-inflammatory effect of Chinese herbal medicine closely, so the past overview Chinese herbal medicine influences the mechanism research of the disease vaginitis immunity reaction progress, Chinese herbal medicine,which influences on the research progress of the inflammatory immunological reactions'mechanism,is summarized, and the composing prescriptions and prescription drugs of Yinlishuang lotion is carring on studying analysis.
The second part of this dissertation concentrated on the study on the quality standard of Yinlishuang lotion. By orthogonal experiment design L9(34),with extractive yield of volatile oil as an index, the optimal oil extraction condition was studied. Three factors were selected including water consumption, soaking time and extracting time. By orthogonal experiment design L9(34), with the content of berberine hydrochloride determined by HPLC as a quanlitative index, the optimal water extracting alcohol precipitating was studied. Four factors were selected including water consumption, extracting times, extracting time and the concentration of alcohol-sediment. The results have showed that the oil extraction condition is adding 10 times water, soaking for 1.5 hours and extracting for 6 hours. And the best water-extraction condition:boiled twice with 10 times water,2 hours for each time and 70% alcohol-sediment.
The last part of this dissertation concentrated on the study on the quality standard of Yinlishuang lotion. Berberine hydrochloride was used as reference substance and trichloromethane-methanol-concentrated ammonia solution(50:10:1) as developer to identify Phellodendron chinense by TLC;Matrine as reference substance and methylbenzene-acetone- methanol(8:3:1) as developer to identify Radix sophorae flavescentis by TLC; Osthole as reference substance and methylbenzene-ethyl acetate- n-hexane (3:3:2) as developer to identify Fructus cnidii by TLC; HPLC was used to determine Berberine hydrochloride in phellodendron chinense,the column was ODS-C18 colunm, the mobile phase was Methyl Cyanides-0.1%phosphoric acid (50:50) (add sodium dodecyl sulfate 0.1g per 100 ml),the detection wavelength was 265mn. The results showed that, Yinlishuang Lotion and the control medicinal material of Phellodendron chinense、Radix sophorae flavescentis and Fructus cnidii with respectively check against an article and check against the position department that the reference substance, respectively same spot by TLC-identification, and the negative control solution have noninterference. The regression equation is Y= 4342774X+7252, r=1.0000 for berberine hydrochloride, and the linear range was (0.0908-2.2700)μg, the average recovery was99.8%,SD and RSD are 1.2%(n=6).
引文
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